Targeting multiple hallmarks of mammalian aging with combinations of interventions

Aging is currently viewed as a result of multiple biological processes that manifest themselves independently, reinforce each other and in their totality lead to the aged phenotype. Genetic and pharmaceutical approaches targeting specific underlying causes of aging have been used to extend the lifespan and healthspan of model organisms ranging from yeast to mammals. However, most interventions display only a modest benefit. This outcome is to be expected if we consider that even if one aging process is successfully treated, other aging pathways may remain intact. Hence solving the problem of aging may require targeting not one but many of its underlying causes at once. Here we review the challenges and successes of combination therapies aimed at increasing the lifespan of mammals and propose novel directions for their development. We conclude that both additive and synergistic effects on mammalian lifespan can be achieved by combining interventions that target the same or different hallmarks of aging. However, the number of studies in which multiple hallmarks were targeted simultaneously is surprisingly limited. We argue that this approach is as promising as it is understudied.


INTRODUCTION
Aging is a time-related process that results in health deterioration and an exponential increase in mortality rate [1].Contemporary geroscience is focused both on investigating the underlying causes of aging and discovering interventions that can prolong animal lifespan.Among mammals, mice are by far the most popular model organism of aging research; however, the attempts to prolong murine lifespan have so far produced only modest results.
The maximum known life extension of mice, resulting from a single intervention, does not exceed 50% (Snell mice with Pit1 knockout or Ames mice with Prop1 knockout).Even in these cases the lifespan of mice is much lower than that of similarly sized mammals with negligible senescence such as the naked mole-rat Heterocephalus glaber [2], indicating that none of these interventions was sufficient to stop aging.One possible explanation is that even if one underlying cause of aging is countered, the remaining aging processes will still limit the animal's lifespan.Thus, we propose the AGING hypothesis that combination therapies can be more efficient against aging.
Combination therapy involves two or more interventions or therapeutic agents that can act synergistically, additively or reduce the side effects of one another.This principle has become the gold standard in various fields of medicine including HIV antiviral therapies [3], cancer treatment [4] and overcoming the evolution of drug resistance [5] such as bacterial resistance to antibiotics [6].
Targeting multiple pathways at once can provide synergistic effects that are expected to be greater than the simple sum of independent effects.For example, chemotherapy kills cancer cells leading to proliferation of cancer-targeting T cells.However, some cancers evolve adaptations that suppress this immune response.Checkpoint inhibitors can lift this suppression allowing T cells to be more effective.Several clinical trials have revealed that chemotherapy works better when combined with this form of immunotherapy [7,8].
Similar examples can be found in the field of aging research.Using the model organism C. elegans Chen and Lahav have shown that a ribosomal protein S6 kinase beta deletion allele, daf-2 loss-of-function allele and their combined effects increase the worm's lifespan by 20%, 168.8% and 454.4% respectively [9].Castillo-Quan et al. have shown that a combination of trametinib, rapamycin and lithium increase the longevity of Drosophila more than each single intervention or pairs of interventions.These drugs inhibit mitogenactivated protein kinase kinase, mTOR complex 1 and glycogen synthase kinase-3 respectively, thus targeting various components of the nutrient-sensing network [10].Recently Kaur et al. showed a synergistic effect of cyclically induced expression of Yamanaka factors (Oct4, Klf4, Sox2, c-Myc) and senolytic peptide (FOXO4-DRI) for lifespan extension in Drosophila [11].Generally, as aging-related pathologies are typically comorbid, targeting multiple biological processes or their separated nodes may be more effective than targeting a single one.
Another rationale for the use of combination therapies is that two treatments can improve each other's effects on a single process.For example, senolytics dasatinib and quercetin have different cell-type specificity and are used together as a cocktail to improve senescent cell clearance [12].A third rationale is to use a treatment that reduces the negative side effects of another.Early experiments on lifespan extension via increased telomerase reverse transcriptase (TERT) activity involved mice with cancer resistant genetic backgrounds with enhanced expression of the tumor suppressors p53, p16, and p19 ARF to counter the supposed oncogenic effect of telomerase [13].Although further studies did not find evidence of increased cancer rates due to telomerase gene therapy and lifespan extension by telomerase alone was of comparable magnitude in regular mice [14,15].Currently, the most comprehensive analysis of synergistic anti-aging interactions is provided by the SynergyAge database [16] which contains the current state of the art collection of data on long-lived and short-lived genetic mutants with over 1800 gene combinations.However, the database does not cover pharmacological and gene therapy interventions which are arguably more relevant for practical human lifespan extending applications.
Here we review existing data on combinations of pharmacological and genetic interventions targeting one or many pathological processes described as the hallmarks of aging in mice [17,18].While we also discuss studies performed on other mammals, we focus on mice because they were used in the largest number of longevity intervention studies.There are important limitations for using mice as a model, given their short lifespan and high cancer mortality.Other models such as dogs are gaining increased attention in geroscience [19], however longevity studies in dogs are still relatively few.
Our ultimate goal is to design and propose several combination therapies that can be tested in mice and in the case of their success translated into human trials, providing a proof-of-principle for this approach.

Methodological considerations
While our fundamental understanding of the underlying causes of aging is still incomplete [20], for the purpose of this review we will consider aging as a complex pathological phenomenon that is characterized by several hallmarks.This is practically useful, because the hallmarks of aging are specifically targetable by known interventions.Up to date the most reliable benchmark for determining the effectiveness of anti-aging interventions is increased lifespan.Thus, our review focuses on lifespan extending interventions; however, we provide a separate analysis for the outcomes of combined therapies on other aging-related conditions.
As of 2023 the following twelve hallmarks of aging (HAs) have been proposed [21]: genomic instability (GI), telomere attrition (TA), epigenetic alterations (EA), loss of proteostasis (LP), disabled macroautophagy (DM), deregulated nutrient-sensing (DNS), mitochondrial dysfunction (MitD), cellular senescence (CS), AGING stem cell exhaustion (SCE), altered intercellular communication (AIC), chronic inflammation (Inf) and dysbiosis (Dys).This is an expansion of the previous list published in 2013 by Lopez-Otin et al. [17].Some authors suggested that additional hallmarks should be considered such as splicing dysregulation (SD), and altered mechanical properties (AMP), which includes stochastic non-enzymatic modification of longlived macromolecules [18,22].Additionally, specific aging contributors such as thymic involution and immune system decline have been highlighted, which we will include in an additional hallmark called immunaging (IA) [23] (Box 1).These additional hallmarks are added because they appear in the scientific literature, and can also be specifically targeted by anti-aging interventions.The hallmarks for which a known targeted life-prolonging therapy exists in mammals, are marked with (+) otherwise with (−).
Interventions designed to target some of the hallmarks of aging have resulted in life extension of various model organisms.Table 1 provides examples of prominent longevity studies that can be linked with a specific aging hallmark.When attributing hallmarks of aging to a given intervention we considered only the primary processes affected without taking into account all possible secondary effects (e.g., the increase of telomerase activity can reduce stem cell exhaustion and the removal of senescent cells can reduce inflammation).Nevertheless, we still assume that one intervention can target multiple hallmarks of aging.
We were able to identify at least one monotherapy that extends the lifespan of normal non-progeroid model mammals for each hallmark of aging except splicing dysregulation and dysbiosis.Although there are no known drugs that extend lifespan by selectively removing modified long-lived macromolecules such as glycated collagen which cause altered mechanical properties of tissues, we speculate that this hallmark of aging can be improved by drugs that lower blood glucose levels such as acarbose or metformin.For example, patients with diabetes that take metformin have lower levels of glycated hemoglobin [24].It is possible that the drug protects other proteins as well.
For dysbiosis we identified one study that found a small increase in lifespan for germ-free mice kept in sterile conditions from birth when compared to mice reared in more conventional settings.This benefit was not present if both groups of mice were food-restricted [25].Another study found that fecal transplants from wild-type mice increased the lifespan in two progeroid mouse models [26].There is also limited evidence for lifespan promoting effects of certain probiotics [27].
The design of combined anti-aging therapies should take into account the possible adverse effects of suggested interventions.For example, rapamycin is known for its immunosuppressive properties.Patients taking therapeutic doses of rapamycin have experienced stomatitis, impaired wound healing, thrombocytopenia, and increased levels of serum triglycerides and cholesterol [28].Doses used to extend the lifespan in mice do not usually result in these side effects; however, impaired glucose homeostasis, gonadal atrophy, and increased incidence of cataracts have been reported [29].Metformin use can lead to vitamin B12 deficiency, gastrointestinal symptoms and other health problems [30].Dasatinib use for cancer treatment is linked to pulmonary arterial hypertension and pleural effusion, platelet dysfunction, and a number of gastrointestinal side effects including intestinal bleeding, although the use of dasatinib as a senolytic may involve lower and thus safer dosages of the drug [31].The loss of function mutations in PROP1 and PIT1 genes of long-lived Ames and Snell mice cause delayed growth and dwarfism.The loss of function of these genes in humans causes severe developmental abnormalities [32,33] and appear not to increase lifespan [34].Finally, most gene therapies result in a concomitant burden on the liver and immune system [35,36] and in some cases, the use of certain vectors is limited due to insertional mutagenesis [37,38].
The existence of adverse effects for potential anti-aging treatments is especially relevant for healthy individuals, because of the increased risk to benefit ratio.Certain adverse effects might be more acceptable in standard pathology treatment because they are less severe than the disease itself, although we should keep in mind that the adverse effects of aging include increased risk of cancer, cardiovascular diseases, chronic obstructive pulmonary disease, cataracts, severe disability and Abbreviations: GI: genomic instability; TA: telomere attrition; EA: epigenetic alterations; LP: loss of proteostasis; DNS: deregulated nutrient-sensing; Mitd: mitochondrial dysfunction; CS: cellular senescence; SCE: stem cell exhaustion; AIC: altered intercellular communication; DM: disabled macroautophagy; Inf: inflammation; IA: immunaging; AMP: altered mechanical properties.Lifespan extension values deduced from provided mortality curves are marked with ( * ).N.A. is not applicable; n.s. is not statistically significant (mentioned as in article).One important limitation of this approach is that sometimes the attribution of hallmarks to an intervention is based on incomplete knowledge.When possible, we tried to rely on the existing consensus about the primary effects of the intervention, however in some instances we had to rely on the molecular mechanisms suggested by the authors that tested the intervention, even though the mechanism was not definitively proven or widely accepted.AGING death.Nevertheless, we must admit that most interventions from Table 1 have not been carefully studied for adverse-effects, which is an important limitation for designing combination therapies.Notably, the adverse effects of some proposed treatments can be potentially offset (or exacerbated) by other components of the combined therapy.For example, immune system depletion can be theoretically offset by bone marrow transplantations and FGF21 inhibition of thymus involution.

Combinations that target different hallmarks of aging
The majority of combination therapy studies in mammals focused on complementing therapies that targeted the same hallmark or set of hallmarks of aging (see Table 2).There are only a few exceptions.First, the combination of TP53, p16 and TERT enhancing mutations demonstrated a strong synergistic effect on the lifespan extension [13].Initial targeting of only two HAs (GI and TA) made mice more resistant to cancer and telomere loss, improved their physical condition, and slowed down the development of aging-associated pathologies and frailty.
Second, a combination gene therapy consisting of three longevity-associated AAV-delivered genes, namely fibroblast growth factor 21 (FGF21), αKlotho and a soluble form of mouse transforming growth factor-β receptor 2 (sTGFβR2) was shown to be beneficial on several mouse models of age-related diseases, such as obesity, type II diabetes, heart failure, and renal failure.Interestingly, the therapy action was synergistic for TGFβ+FGF21 and TGFβ+αKlotho, while the effect was negative for FGF21+αKlotho (the combination worked worse than the individual intervention results for all 4 diseases) [82].The components of the therapy target different signaling pathways and can be related to the AMP and AIC hallmarks.The triple combination did not outperform either of the two best double combinations by its effects on age-related pathologies.
Unfortunately, the effects of these therapies on lifespan were not reported [82].

Combinations based on rapamycin
Among the pharmacological combination therapies that targeted the same HAs many involved the mTOR inhibitor rapamycin in conjunction with other molecules that affect the DNS hallmark, such as, the antidiabetic inhibitor of liver glucose production metformin, and inhibitor of α-glucosidase acarbose.While metformin and acarbose are readily used as anti-diabetic drugs, chronic rapamycin treatment has been shown to increase glucose intolerance in mice [83,84].This is one potential explanation for the increased benefit of the combination.Although some researchers argue that the diabetes-like condition caused by high doses of rapamycin might actually be beneficial in terms of aging by itself.
Combinations of rapamycin and metformin have resulted in an additive effect on mouse lifespan [47], while combinations of rapamycin and acarbose have led to an additive increase in male lifespan with no additive effect in females [85] (if compared to studies where rapamycin [44] or acarbose [75] were used as a monotherapy).One additional study tested a combination of rapamycin, acarbose and phenylbutyrate [86] (a drug that was previously shown to rescue cognitive impairment in a mouse model for Alzheimer diseases) [87].The combination worked better than individual treatments in decreasing tumor burden as well as cognitive and physical decline in aged mice.Performance tests favored the combination and rapamycin alone comparing to the other two drugs.All drugs reduced the severity of lesions, but the best results were observed for the combination therapy.
Rapamycin and metformin have some overlapping targets and mechanisms of action: mTOR pathway inhibition, enhancing insulin/IGF1 signaling, and autophagy activation.Acarbose affects glucose metabolism by slowing down breakage of polysaccharides to glucose in the intestine.Phenylbutyrate promotes the catabolism of branched-chain amino acids and correspondingly downregulates mTOR [88].

Combinations of drugs used against cardiovascular disease
A combination of the statin simvastatin and the angiotensin-converting enzyme inhibitor ramipril cause a small yet synergistic increase of male mouse lifespan [89].Simvastatin lowers low-density lipoprotein (LDL) via reversible inhibition of HMG CoA reductase.Neither of the drugs was shown to increase normal rodent lifespan [43,101], but they did so in combination.
While the mechanisms of action of these drugs are different, they both target similar HAs, namely altered intercellular communication (AIC).One important side-effect of the statin and combined treatment was the increased occurrence of benign lung tumors and hemorrhagic diathesis, but this did not affect overall survival according to the study [89].
It should be noted, that translating any of these findings to humans is particularly difficult because unlike humans mice rarely develop coronary artery atherosclerosis with age, do not exhibit the same high density lipoprotein (HDL) profiles, and have other important difference that affect their cardiovascular system [102].

Combinations that target senescent cells
The use of senolytics dasatinib + quercetin (D+Q) is another example of the approach where both interventions target the same HAcellular senescence.Quercetin alone does not improve murine lifespan [103].The life-extending properties of dasatinib alone have not been investigated, but it was shown that its combination with quercetin is more efficient in elimination of senescent cells and prolongs the lifespan of mice.The oral administration of dasatinib and quercetin decreased the number of naturally occurring senescent cells and their secretion of frailty-associated proinflammatory cytokines in explants of human adipose tissues.Moreover, in both senescent celltransplanted young mice and naturally aged mice the combination therapy alleviated physical dysfunction and increased post-treatment survival by 36% [104].
Other trials have shown that dasatinib plus quercetin can improve metabolic function, attenuate adipose tissue inflammation and age-dependent intervertebral disc degeneration [105] as well as prevent age-related uterine dysfunction and fibrosis [106].
Dasatinib is a senolytic agent, inhibiting tyrosine kinases, while the antioxidative flavonoid quercetin apart from senolytic activity via inhibition the antiapoptotic protein Bcl-xL is anti-inflammatory [107].
Additionally, the combination of senolytics dasatinib and quercetin was shown to selectively remove senescent cells in a phase 1 study on nine human patients suffering from diabetic kidney disease [108].
Another study on 14 patients with idiopathic pulmonary fibrosis (IPF) showed that intermittent administration of D+Q slightly but significantly improved their physical functions such as grip strength [109].In patients with diabetic kidney disease D+Q treatment decreased senescent cell burden in adipose tissue and epidermis [108].In both human trials a so-called "hit-and-run" administration of senolytics was applied, which is either single dose or intermittent administration.This is rationalized by several reasons: first, senescent phenotypes develop slowly, thus after elimination of senescent cells new ones will not appear soon.Second, the senolytics' mechanism of action does not require them to be continuously present.Finally, such a regimen was shown to reduce the side effects of dasatinib [110,111].
Another promising approach to remove senescent cells was demonstrated on a mouse model by applying adoptive infusion of senolytic natural killer (NK) cells boosted by acein, a dopamine-releasing peptide, which caused no senolytic effect by itself.The therapy resulted in reduced local and systemic senescence-associated secretory phenotype (SASP) in aged mice and enhanced the elimination of senescent cells compared to NK cell infusion alone [112].

Combinations based on calorie restriction
Calorie restriction (CR) is one of the most studied methods of life extension in mice, rats and other mammals [113].The mechanism of lifespan extension through CR is usually attributed to a number of pathways that involve IGF1/FOXO [114], mTOR [115], SIRT1 [116] and other actors.Mice with genetically determined dwarfism (GH, GHR, GHRHR, PROP1 mutations that suppress the GH/IGF1 axis) are well known for their increased lifespan, and there is a number of studies in which such mice were fed with calorie-restricted diets.In most cases such combinations extended the animal's lifespan even further.For example, PROP1 deficiency alone extended lifespan by 37%, and in combination with CR by up to 69% compared with wild-type ad libitum fed controls [96].
The potential mechanism of lifespan extension in mice by PROP1 deficiency and by CR could be mediated by increased insulin sensitivity, which is further improved by this combination [98].
In rats suppression of GH also extended lifespan, and calorie restriction enhanced this effect [100].This effect appears to be additive rather than synergistic.The main exceptions are GHR knockout mice that are less responsive to diet restrictions.CR failed to increase overall, median, or average lifespan in these mice and increased maximum lifespan only in females and to a rather small extent (9%) [117].Also, the combination of GHR KO and CR does not improve insulin sensitivity more than the separate interventions [98].AGING

Combinations based on partial epigenetic reprogramming
The genes Oct4, Sox2, Klf4 and c-Myc are the four Yamanaka factors (OSKM) that can be used in combination to reprogram specialized cells such as fibroblasts into induced pluripotent cells [118,119].Short-term activation of these genes can be used for partial epigenetic reprogramming that does not lead to complete loss of cellular identity but reduces the biological age of targeted cells as measured by epigenetic clocks and other markers of aging [120].We can classify each OSKM component as aiming for the EA and SCE hallmarks of aging, thus the targets of this therapy overlap.
Virus delivery of the OSK subset of Yamanaka factors to extremely old mice (124 weeks old) decreased their biological age measured by methylation epigenetic clocks, improved their health (decreased frailty) and extended median lifespan by 6% [95].Another study, which utilized a full set of Yamanaka factors, showed that their long-term partial induction led to the reversion of the epigenetic clock and reduced the expression of numerous genes associated with inflammation, senescence, and stress response, resulting in improved tissue rejuvenation.The phenotype changes were detectable in different tissues, such as the kidney and skin.Epigenetic clock reversal as well as metabolic and transcriptomic changes, including downregulation of pro-inflammatory genes and cellular senescence could be observed after treatment, with the degree of manifestation dependent on the duration of OSKM expression [121].

Combinations of tumor suppressors
Synergistic lifespan extension can be achieved not just by targeting the same HAs but the same biological processes, e.g., elevated expression of oncosupressors p53 and Arf extended mice median lifespan 16%, while p53 alone only decreased tumor incidence, and Arf had a very weak effect on lifespan [93].The addition of a TERT transgene extended the lifespan of p53/Arf over-expressing mice by 40%.While p53 and Arf alone protects mice from cancer, neuromuscular degeneration and oxidative damage, addition of TERT attenuated the accumulation of histone markers of double-strand breaks, particularly at telomeric regions, and reduced inflammation [13].

Combinations that target oxidative stress
According to the free-radical theory of aging, lifespan can be increased by exogenous administration of antioxidants or molecules that enhance the organism's ability to remove notable oxidants such as H2O2 [122].This theory has a number of problems, including observations that certain antioxidants including vitamins A and E may actually increase mortality [123].One possible explanation is that the introduction of additional antioxidants may negatively affect the endogenous antioxidant systems.Nevertheless, a number of studies tested combinations of interventions aimed at reducing oxidative stress.
One such intervention targeted a single HA (MitD) via overexpression of peroxisomal catalase and superoxide dismutase (PCAT and SOD1 genes) had the synergistic effect on the median but not maximal lifespan [94].Another study examined the effects of Cu and Zn superoxide dismutase overexpression separately or together, but none of the interventions extended the lifespan of mice [124], contrasting the results of similar experiments on Drosophila [125].
Antioxidants are often used to combat aging-related diseases, but there are examples where pro-oxidants provide similar effects.A pro-oxidant combination of chelated copper and resveratrol creates free oxygen radicals which eliminate cell-free chromatin particles originating from dead cell debris that can cause inflammation.Thus, in this case pro-oxidants are antiinflammatory.Application of the treatment to aged mice led to a reduction of several hallmarks of aging in brain cells including TA, GI, Inf, CS and MitD.The therapy also resulted in significant downregulation of glucose blood levels, cholesterol, amyloid deposition, and C-reactive protein [126].
Another study reported that a combination therapy of glycine and N-acetylcysteine (Gly-NAC) given together significantly prolonged the median and maximum lifespan of mice by 23.7% and 33.1% respectively [91].Both compounds are claimed to act through ameliorating the age-associated glutathione deficiency and reducing oxidative stress.Glycine itself was previously shown to have only a very modest (4-6%) effect on lifespan [81] while N-acetylcysteine had no effect on female lifespan, but provided a 44% increase in median lifespan in males [127].We noticed that in the N-acetylcysteine study male mortality appears to be unusually high in the control group, so we doubt that single N-acetylcysteine provided lifespan increase.
Various natural products with presumed antioxidant and anti-inflammatory properties are considered as potential geroprotectors, however many of them do not show any pronounced effects on murine lifespan.The combination of pycnogenol, quercetin and taxifolin, did not expand lifespan of the experimental animals.The same conclusion was made for single treatments of   blueberry, pomegranate, green and black tea, cinnamon, sesame, French maritime pine bark, green tea extract, black tea extract and morin.The authors suggest a number of previous reports that these natural products expanding longevity may have resulted from induced caloric restriction [128].

Combinations that were studied for anti-aging effects other than lifespan extension
Some studies of combination therapies were designed to test if they can reverse separate aging-related phenotypes and/or pathologies without attempting to register any effect on general lifespan.
One combination gene therapy against type II diabetes was aimed at overexpressing the transgenic EZH2 protein, a key component of the Polycomb complex, while simultaneously knocking down the MII1 gene, a part of the Trithorax complex.It resulted in successful repression of the Ink4a locus and rejuvenation of the pancreatic islets through promotion of β-cell proliferation.Notably, the EZH2 alone was not sufficient to cause improvements in mice older than 8 months while simultaneous MII1 knockdown acted well even in aged animals.Additionally, a combination of EZH2 overexpression and JnjD3 knockdown in beta cells of aged mice enhanced replication of beta cells, unlike the separate interventions [130].
There is also limited evidence that some combination therapies can ameliorate age-related phenotypes in humans.For example, a combination of growth hormone (GH), dehydroepiandrosterone (DHEA) and metformin was used on a group of 10 patients aged 51-65 years.GH was aimed at thymus regeneration and reduction of immunosenescence, while two other drugs were supposed to reduce the diabetogenic side effects.Indeed, the therapy resulted in regenerative response in thymus and bone marrow and delayed epigenetic clock advancement in the studied tissues [131].
A prominent study used a combination of three Yamanaka factors (excluding the carcinogenic c-Myc) to regenerate damaged optic nerves and recover vision in mice with glaucoma served epigenetic age reversal.
No pronounced side effects were observed [132].

Effect of combination therapies on lifespan of progeroid and disease animal models
Although our review focuses mainly on experiments where therapeutic interventions were able to significantly expand the lifespan of normally aging lab mice, we decided to make a separate overview of combination AGING therapies which prolonged lifespan and healthspan in progeroid and other rapidly aging mouse models.Despite progeroid models being less representative of the normal aging processes, studies on them still remain an important source of knowledge about the mechanisms of aging and anti-aging treatments [133].
In a well-known Hutchinson-Gilford Zmpste24 −/− progeria mouse model of premature aging, a combination of three synergistically acting drugs targeting the AMP hallmark (by preventing vascular calcification) has shown a significant though moderate median lifespan increase.The application of extracellular ATP yielding pyrophosphate (which is a known inhibitor of calcium-phosphate deposition and therefore of vascular calcification [134]) was boosted by two drugs: tissue-nonspecific alkaline phosphatase (TNAP) inhibitor levamisole and ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor ARL67156.The drugs enhanced the production of pyrophosphate from ATP and reduced its hydrolysis, respectively, leading to prevention of vascular calcification.Notably, ATP alone did not play any effect on the lifespan of studied animals [135].
A combined treatment with statins (pravastatin) and aminobisphosphonates (zolendronate) was enough to synergistically extend longevity in the same mouse model.Both drugs target the same hallmark of aging, namely the loss of proteostasis, by simultaneously inhibiting two interchangeable pathways of prelamin A and progerin prenylation.The therapy significantly prolonged median and maximum lifespan (Table 4) and evidently improved the aging-like phenotypes typical for the model, including growth retardation, loss of weight, lipodystrophy, hair loss and accumulation of bone defects [136].
Another progeroid mouse model was efficiently treated by the combination of Yamanaka factors (OSKM).LAKI mice carrying a truncating lamin mutation were subjected to short-term cyclic OSKM induction, which resulted in amelioration of cellular and physiological hallmarks of aging and led to an extension in median and maximal (up to 20%) lifespan [137].
Autoimmunity is a well-known age-dependent pathology.Studies of autoimmune responses often involve the autoimmunity prone NZB × NZW F1 mice model.On this model the additive effect of calorie restriction and fish oil, targeting the DNS and INF hallmarks was observed.The combination was shown to reduce levels of several important proinflammatory factors, including TNF-α and NF-kB and to upregulate antioxidant pathways leading to a dramatic median lifespan increase of 166% [138].Another experiment on the same model involved mice treatment with a known immunosuppressant cyclophosphamide along with an immunostimulator tilorone.This combination of drugs exhibited an additive effect on lifespan extension [139].

Developing combination therapies to enhance mammalian longevity
It appears that combination therapy is a promising approach for mammalian lifespan extension.It can work when targeted hallmarks of aging are different and even in cases when the presumed hallmarks overlap.However, the number of tested combinations of longevity-enhancing treatments is relatively small and most studies are done in rodents.The design and implementation of additional studies is warranted, including those in other species of mammals.
The current availability and diversity of lifespan promoting therapies allows the possibility to design studies in mice targeting at least thirteen hallmarks of aging simultaneously.Nine of these hallmarks can be readily targeted in animals of any genetic background.For example, this can be accomplished with a combination of dasatinib plus quercetin, FGF-21, acarbose, methionine restriction, young bone marrow transplantation and gene therapy for VEGF and TERT activation and hypothalamus-restricted Nf-Kb inactivation (example combination 1, Figure 1).Three more hallmarks can be targeted by genetically engineering mice overexpressing SIRT6 and by creating mclk1 knockout heterozygotes (example combination 2, Figure 1).Theoretically the same effects can be produced by experimental gene therapies.However, example combinations 1 and 2 involve surgical procedures that by themselves can be traumatizing, a lentiviral gene therapy that can cause insertional mutagenesis, a CMV gene therapy that in theory may contribute to immune system depletion, an AAV gene therapy that like the other two gene therapies may cause immune reactions and liver toxicity.
Given that there are many possible interventions that have provided lifespan extension in mice as single therapies, for the conservation of effort and resources, it would be reasonable to develop strategies for prioritizing the most promising combined therapies.Ideally, these strategies should take into account the cost and effectiveness of individual treatments, their theoretical interactions and whether the targeted hallmarks overlap.We would also argue that we should prioritize interventions that can be applied to already aged animals given the ultimate practical goal of prolonging the lifespan of existing humans.Perhaps interventions that are evidently less effective in humans than in mice, dangerous or difficult to sustain should be deprioritized for practical purposes.For example, calorie restriction without malnutrition while being highly effective at extending mouse lifespan is unlikely to provide proportional life extension in humans and might be difficult to follow [144].Indeed, it is currently unclear whether calorie restriction in the true sense (i.e., not rescue from overeating/obesity) has any of the effects seen in rodents when it comes to humans.Taking these considerations into account, one strategy could be to combine therapies that provided the highest lifespan extension as individual treatments.In descending order these would be (VEGF gene therapy up to +49% median), methionine restriction (up to +42% mean), TERT gene therapy (up to +41% median), follistatin gene therapy (+32% mean) and bone marrow transplantation (up to +28% mean).Note that the provided value of increased lifespan expectancy for bone marrow transplantation might be inflated given the high mortality in one of the studies' control groups.
A more conservative result of +12% median lifespan increase was reported in a more recent study [64].
Another strategy is to select the smallest number of treatments that cover the largest number of aging hallmarks (while still preferring treatments with highest lifespan extension values).For example, dasatinib + quercetin (CS + inf), rapamycin (DM + LP + DNS), VEGF gene therapy (AIC), bone marrow transplantation (SCE) and telomerase gene therapy (TA), perhaps with the addition of acarbose.Unfortunately, as mentioned earlier, we were unable to identify interventions that have confirmed positive effects on lifespan and can be applied to adult animals for several hallmarks of aging.
A third strategy is to start with the best existing combinations and expand them with interventions that provide the highest lifespan extension as an individual treatment but were not used before, preferably targeting a hallmark of aging that the initial combination did not.In this case we would recommend starting with rapamycin + acarbose (up to +37% lifespan extension) followed by the individual combinations from the first strategy.
A fourth strategy may be based on systematic screening for the synergistically best pairs of combinations that target different hallmarks of aging.This approach might also elucidate conflicting impacts of interventions that can offset or even worsen each other's effects.The absence of an additive and synergistic effect may indicate, in particular, that the downstream effects of two therapies overlap.
Finally, a fifth strategy might consider investigating combinations that consist of widely-used molecular components with well-documented safety profiles in humans.Such combinations might include acarbose, glycine, taurine, and spermidine.
It's worth noting that some combinations might be considered more practically feasible in real-life clinical settings.For example, small molecule drugs and supplements are easier to use than gene therapies, followed by bone-marrow transplantation.However, the widespread use of gene therapies by the general public is possible, given the availability of COVID vaccines which are similar in design.Multiple gene therapies have successfully finished human clinical trials and are available for a wide range of diseases from hemophilia A and B, to RPE65-mutation-associated retinal dystrophy, dystrophic epidermolysis bullosa and certain types of cancer [145].Given the large amount of genes potentially involved in aging such as those reviewed in comprehensive Open genes database [146], we believe gene therapies are desirable and viable components of future anti-aging combination therapies.

Further directions
We observe that a number of genetic mutations increase the lifespan of mammals, but the effects of these mutations were not tested as possible longevity enhancing gene therapies.This includes gene therapies to overexpress Sirt6, Sirt1, p53 (+ p16 + Arf), FoxM1, catalase OE in peroxisomes + superoxide dismutase and the reduction of expression of GH, GHR, GHRHR, Prop1, mclk, macrophage inhibitory factor, IGF1R and Becn1F121A/F121A mutation in beclin 1.Individual testing of these approaches could provide novel single interventions that could be combined with future effective combination therapies.The combination of FGF21, αKlotho and sTGFβR2 deserves deeper investigation in terms of lifespan prolongation as a promising combination gene therapy that uses a number of genes that were all previously described as healthspan-or lifespan-promoting [51,71,147,148].
There are some genetic and pharmacological interventions that are promising but, to our surprise, have not been tested in mice.One of them is the upregulation of FOXO3a, which is linked to human longevity and healthspan [149][150][151][152].Some existing FOXO3a gene variants are associated with extreme longevity in human populations [149].It is also known to be a positive regulator of healthspan, that affects the aging of the cardiovascular system [153].The lifespan-expanding function of FoxO proteins is highly evolutionary conserved: the single FoxO homolog in Hydra contributes to the anti-aging mechanisms of this freshwater polyp [154,155].The prolongevity effects of homologous genes have been vastly studied in C. elegans, where they are associated with the DNS HA [156].FOXO3 mediates the life-extending effect of CR in mice [114], but we haven't found any studies where it was specifically overexpressed in mammals and lifespan changes were assessed.

Limitations
One important limitation of our analysis is that the effects of treatments on lifespan or other aging-related AGING metrics in mice are at least to some extent dependent upon the conditions in which the animals are maintained and the genetic background of the strains used.Long-term breeding in laboratories has led to some strains being mildly progeroid, thus some of the life-extending effects could result from the rescue of the underlying conditions [157,158].This may further reduce the translatability of pro-longevity treatments in mice to other animals including humans.Additional studies in other species, such as dogs, could help elucidate the pro-longevity treatments that are more generalizable.
Another limitation comes from the hallmarks of aging.While currently we do not have a better framework for describing the aging process, the hallmarks of aging have relatively limited explanatory value regarding the general etiologies of aging and do not discern the primary and secondary causes of aging [20].As we understand more about the aging process, we will be able to provide a better framework for selecting the most efficacious combination treatments.
There is also the question whether aging can truly be intervened.We believe it is possible given that the lifespans of many model organisms ranging from roundworms to mice have been greatly improved.The potential for human life extension is also supported by the increase of human lifespan during the course of evolution, when compared to our ancient ancestors.Finally, the existence of negligible senescence in some mammals, such as the naked mole rat suggests that aging is not an insurmountable challenge for life [2].

CONCLUSIONS
Aging is a complex problem that most likely requires multiple simultaneous solutions.Several combined therapies that target multiple different hallmarks of aging or even the same hallmarks of aging have been shown to provide additive and sometimes synergistic effects on mammalian lifespan.Nevertheless, most tested combined therapies involved targeting of only a small fraction of aging hallmarks and many potentially beneficial combinations have not been tested.We propose several strategies to select potential combination therapies against aging.These strategies include the idea of targeting as many hallmarks of aging as possible, prioritizing single interventions that provide the highest increase in lifespan, or adding single interventions to existing combinations with highest contributions to lifespan.We believe that the use of combination therapy is not only feasible in the field of longevity but provides the best practical opportunity to prevent age-related pathologies and enhance human lifespan in the nearest future.

Figure 1 .
Figure 1.Possible strategies of development of aging combination therapies.This figure illustrates the various hallmarks of aging that could be addressed by different existing interventions.Additionally, it proposes four example combinations of therapies, each detailing which specific treatment targets a particular hallmark of aging.